Viruses are a heterogeneous group of intracellular infectious agents that depend in varying degrees on the host synthetic machinery for replication. The poxviruses are large, double-stranded DNA viruses that are assembled in the cytoplasm of infected cells involving complex replication mechanisms (Moss, 2007). Attachment, internalization, and disassembling of poxviruses precedes the initiation of three waves of mRNA synthesis. The early wave codes for virus growth factors and decoy cytokine receptors. Decoy receptors for both type I and type II interferons (IFNs) are produced during early protein synthesis in poxvirus infected cells, thus blunting perhaps the most important innate host defense system against viral infections (Moss and Shisler, 2001). A well-known example of this is the B8R protein of vaccinia virus, which is a homolog of the extracellular domain of the IFNγ receptor (Moss, 2007).
Encephalomyocarditis (EMC) virus is a small single-stranded RNA picornavirus of the plus strand orientation with wide host range (Racaniello, 2007). In mice, EMC virus infection is lethal, but is quite susceptible to IFNγ or an IFNγ mimetic treatment at early stages of infection (Mujtaba et al., 2006). The IFNγ mimetic is also effective against vaccinia virus infection even in the presence of B8R decoy receptor (Ahmed et al., 2005; Ahmed et al., 2007). The IFNγ mimetic is a small peptide corresponding to the C-terminus of IFNγ that functions intracellularly and thus does not interact with the extracellular domain of the IFNγ receptor (Ahmed et al., 2005).
The IFNγ mimetic is also effective against another large double-stranded DNA virus called herpes simplex 1 or HSV-1 that replicates in the cell nucleus (Frey et al., 2009). Close relatives include the herpes Zoster virus and cytomegalovirus (Roizman et al., 2007). The broad spectrum of antiviral activity of IFNγ mimetics is unique in that we are unaware of any other small antiviral that exhibits strong activity against poxviruses, picornaviruses, and herpes viruses.
The IFN system is regulated by an inducible endogenous tyrosine kinase inhibitor called suppressor of cytokine signaling 1 or SOCS-1 (Yoshimura et al., 2007; Mansell et al., 2006; Yasukawa et al., 1999; Kobayashi et al., 2006; Croker et al., 2008). SOCS-1 is a member of a family of inducible proteins that negatively regulate IFN and other cytokine signaling via inhibition of JAK/STAT signaling (Yoshimura et al., 2007). There are currently eight members of the SOCS family, SOCS-1 to SOCS-7 and cytokine-inducible SH2 protein. SOCS-1 has distinct regions or domains that define the mechanism by which it inhibits the function of JAK tyrosine kinases such as JAK2 that are involved in activation of STAT transcription factors (Yoshimura et al., 2007). The N-terminus of SOCS-1 contains a SH2 domain, and N-terminal to it is an extended SH2 sequence (ESS) adjacent to a kinase inhibitory region (KIR) (Yoshimura et al., 2007). These domains or regions of SOCS-1 bind to the activation and catalytic regions of JAK2 and block its function. The C-terminus of SOCS-1 contains a domain called the SOCS box, which is involved in proteasomal degradation of JAK2. It has been shown that the KIR sequence of SOCS-1 binds to a peptide corresponding to the activation loop of JAK2, pJAK2(1001-1013), and that the peptide pJAK2(1001-1013) blocked SOCS-1 activity in cells (Waiboci et al., 2007). Specifically, pJAK2(1001-1013) enhances suboptimal IFN activity, blocks SOCS-1 induced inhibition of STAT3 activation, enhances IFNγ activation site promoter activity, and enhances antigen-specific proliferation.
Influenza A virus is a segmented negative strand RNA virus that is responsible for over 30,000 deaths annually in the United States (Palese and Shaw, 2007). Pandemic influenza A virus infection can cause the deaths of millions world-wide. Type I IFNs are an important early innate immune response cytokine against influenza respiratory infections (Szretter et al., 2009). Influenza virus-encoded nonstructural protein NS1 is multifunctional and is important in virus defense against IFNs by a mechanism(s) that is not fully understood but may involve induction of SOCS-1 and SOCS-3, which in turn would negatively regulate IFN signaling (Pothlichet et al., 2008).
Herpes Simplex Virus (HSV) is a member of a broad class of double-stranded DNA viruses that undergo replication in the cell nucleus. Examples of other members are varicella-zoster virus (VZV) and cytomegalovirus (CMV) (Roizman et al., 2007). It is estimated that HSV-1 infects 60 to 80 percent of the people throughout the world, and persists for life in the infected individuals (Diefenbach et al., 2008; Koelle and Corey, 2008; Cunningham et al., 2006). Primary infection commonly occurs through cells of the mucous membrane and is often asymptomatic. This is followed by uptake of virus by sensory nerve fibers and retrograde transport to the cell body of the neurons in the dorsal root or trigeminal ganglion. Here, acute infection is converted to latency and from which HSV-1 periodically migrates down the nerve tissue to again infect mucosal cells for overt disease (Roizman et al., 2007; Diefenbach et al., 2008; Koelle and Corey, 2008; Cunningham et al., 2006).
HSV-1 infection is characterized by a strong cytokine response in infected cells, particularly the induction of type I IFNs (Cunningham et al., 2006). Infection of keratinocytes, for example, results in induction of large amounts of IFNα and IFNβ as well as interleukins 1, 6, and β-chemokines (Mikloska et al., 1998). IFNs, macrophages, natural killer (NK) cells, and gamma/delta T cells all play an important role in host innate immune response to HSV-1 (Cunningham et al., 2006). Toll-like receptor (TLR) 2 is activated on the cell surface by HSV-1, while TLR-9 is activated intracellularly by viral DNA. The latter stimulus is thought to play an important role in induction of IFNα by HSV-1 (Cunningham et al., 2006).
The adaptive immune response plays an important role in confining HSV-1 and other herpes virus infections to a latent state where CD8+ T cells and IFNγ play critical roles (Knickelbein et al., 2008; Sheridan et al., 2007; Decman et al., 2005). It is functionally connected to the innate immune system where NK cells can serve as a source of IFNγ, which is also produced by CD4+ and CD 8+ T cells. IFNγ can exert direct antiviral activity as well as induce upregulation of MHC class I and class II molecules on macrophages, dendritic cells, and keratinocytes (Decman et al., 2005). Direct effects of IFNγ as per a mouse model suggest that this IFN prevents reactivation of HSV by inhibition of function of the key intermediate protein ICP0 (Mossman, 2005). Interaction of the antigen presenting cells with CD4+ T cells induces CD8+ T cells to control HSV-1 levels in mucosal lesions (Arduino and Porter, 2008; Patel et al., 2007).
HSV-1 has developed several mechanisms to inhibit both the innate and adaptive immune responses to infection. HSV-1 downregulation of class I MHC expression occurs through high affinity binding of viral immediate early gene product ICP47 to the transporter associated with antigen processing (TAP) (Burgos et al., 2006), which blocks IFNγ induction of cytotoxic CD8+ T cells (Goldsmith et al., 1998). IFN signaling is also inhibited by blockage of JAK/STAT transcription factor phosphorylation by an unknown mechanism (Chee and Roizman, 2004). ICP0 is thought to enhance proteasome-dependent degradation of IFN stimulated genes (ISGs) (Halford et al., 2006; Edison et al., 2002). A recent study suggests that HSV-1 can exert an anti-interferon effect by activation of a protein called suppressor of cytokine signaling 3 (SOCS-3) (Yokota et al., 2004).
Currently, there are no effective therapeutics available against HSV infection, except the nucleoside analog acyclovir (Dorsky and Crumpacker, 1987), which is known to have serious side effects. A search for a vaccine against HSV has remained elusive because of the successful adaptation to the host used by HSV (Koelle and Corey, 2008). Along with direct effects, infection with HSV has been found to increase the incidence of HIV infection, probably due to HSV-associated lesions (Wald and Link, 2002). Because of this interplay between HSV and HIV, it is conceivable that anti-HSV treatment may reduce the incidence of infection with HIV.
Type I interferons (IFNs), IFNα and IFNβ have been clinically approved for the treatment of hairy cell leukemia, chronic myelogenous leukemia, melanoma, hepatitis C virus infection, and multiple sclerosis. Treatment with these IFNs is associated with severe side effects, including bone marrow suppression, depression, and fever, which has resulted in several patients dropping out of treatment programs. There remains a need in the art for type I IFN mimetics that can provide the same benefits as the parent interferons, while having less of the undesirable effects.
The classical model of cytokine signaling dominates our view of specific gene activation by cytokines such as the interferons (IFNs) (Levy and Darnell, 2002). In this model, ligand activates the cell solely via interaction with the extracellular domain of the receptor complex. This in turn results in the activation of receptor or receptor-associated tyrosine kinases, primarily of the Janus or JAK kinase family, leading to phosphorylation and dimerization of the STAT transcription factors, which then disassociate from the receptor cytoplasmic domain and translocate to the nucleus. This view ascribes no further role to the ligand, JAKs, or the receptor in the signaling process. Further, there is the suggestion that the STAT transcription factors possess intrinsic nuclear localization sequences (NLSs) that are responsible for nuclear translocation of STATs and specific gene activation (McBride et al., 2000; Melen et al., 2001; Begitt et al., 2000).
It has recently been acknowledged that the classical model of JAK/STAT signaling was over-simplified in its original form. In the case of IFNγ, complexity beyond simple JAK/STAT activation is indicated in the relatively recent demonstration that other pathways, including MAP kinase, PI3 kinase, CaM kinase II, NF-κB, and others cooperate with or act in parallel to JAK/STAT signaling to regulate IFNγ effects at the level of gene activation and cell phenotypes (Gough et al., 2008). All of these pathways are generic in the sense that a plethora of cytokines with functions different from those of IFNγ also activate them. Thus, uniqueness of function would seem to depend on cytokine control of complex and unique qualitative, quantitative, and kinetic aspects of activation of these pathways. This uniqueness has thus far not been demonstrated.
At the STAT level, there is evidence of a functional interaction between different STATs in gene activation/suppression, which provides more insight into STAT mediation of cytokine signaling. The induction of IL-17 by activated STAT3, for example, was countered by IL-2 activation of STAT5 (Yang et al., 2011). It was demonstrated by chromatin immunoprecipitation (ChIP) sequencing that STAT3 and STAT5 bound to multiple common sites across the IL-17 gene locus, including non-coding sequences. The activation state of these STATs was not addressed. Induction of STAT5 by IL-2 resulted in more binding of STAT5 and less binding of STAT3 at these sites, whereas induction of STAT3 by IL-6 induced the opposite; the combination of the two STATs resulted in dynamic regulation of the IL-17 gene locus by the opposing effects of IL-2 (STAT5) and IL-6 (STAT3) (Yang et al., 2011). A similar complementarity was observed with STAT4 and STAT6 with respect to Th1 and Th2 cell development, but with much less competition for binding sites at coding and non-coding regions of the gene (Wei et al., 2010). These Yin-Yang interactions of STAT transcriptions factors are referred to as specification with respect to lymphocyte phenotypes. It is not clear, however, as to how these STAT interactions at the level of DNA binding translate into specific gene activation by the inducing cytokine.
There is evidence that JAK kinases, including the mutant JAK2V617F, play an important role in the epigenetics of gene activation in addition to STAT activation in the cytoplasm (Dawson et al., 2009). Leukemic cells with a JAK2V617F gain-of-function mutation have constitutively active JAK2V617F in the nucleus. This leads to phosphorylation of Y41 on histone H3, which results in disassociation of heterochromatin protein 1α, HP1α. The heterochromatin remodeling was associated with exposure of euchromatin for gene activation. Although present in the nucleus, wild-type JAK2 was only activated when K562 cells were treated with PDGF or LIF, or when BaF3 cells were treated with IL-3. The question of how a ligand/receptor interaction resulted in the presence of activated JAK2, pJAK2, in the nucleus was not addressed, nor its targeting mechanism to discrete genomic sites and specific promoters.
It has been shown in the case of IFNγ that receptor subunit IFNGR1 is associated with pJAK2 and phosphorylated histone H3Y41 at the promoter of the IRF1 gene, while the β-actin gene is unaffected, since it is not acted on by IFNγ (Noon-Song et al., 2011). Activated TYK2, pTYK2, in the nucleus and at promoters of genes activated by type I IFNs. TYK2 is also activated by other cytokines such as IL-12 and IL-23, which have biological effects different from IFN (Jones and Vignali, 2011; Duvallet et al., 2011). We were therefore particularly interested in whether there was an association between pTYK2 and type I IFN receptors at the promoters and chromatin of genes activated by these IFNs and whether such association provided insight into pTYK2 induced specific epigenetic events in genes activated by the IFNs. The findings provide insight into the mechanism of specific gene activation by type I IFNs, including the associated epigenetic events.